A common technique for sequencing DNA is the Southern blot. This technique involves transferring DNA fragments from agarose gels to a membrane, typically to a nitrocellulose membrane. The DNA fragment would then be hybridized with labeled complimentary DNA probes. More recently, various nylon membranes have been used in place of nitrocellulose, since nylon membranes generally bind DNA thereto better than nitrocellulose. Furthermore, it has also been found that DNA binding to a nylon or nitrocellulose membrane, could be considerably enhanced if the DNA fragments, following placement on the membrane, were irradiated with ultraviolet light ("UV") at a peak emission of 254 nanometers ("nm"). Such a technique has been described by Kandjian, Biotechnology, Vol. 5, p. 165-167 (Feb., 1987), as well as by Church et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 81, p. 991-995 (Apr., 1984). The enhanced DNA binding to the substrate permits any initial probe/DNA hybrids to be denatured, and the bound DNA fragments reprobed many times with probes of different PG,3 sequences, without any significant loss of signal from any hybrids formed. Thus, the overall sensitivity of the Southern blot technique is enhanced.
It has also been recently known to irradiate DNA with 254 nm UV to form thymine dimers. Thymine dimer formation results in only partial digestion of a DNA specimen upon exposure to a restriction enzyme, and thereby facilitates restriction site mapping of the specimen. Formation of thymine dimers by UV irradiation, with subsequent restriction site mapping, is described by Whittaker et al., Gene, Vol. 41, p. 129-134 (1986), and by Cleaver, Biochimica Et Biophysica Acta, Vol. 697, p. 255-258 (1982). Furthermore, as suggested by Saito et al., Tetrahedron Letters, Vol 22, No. 34, pp. 3265-3268 (1981), 254 nm UV can also been used to irradiate thymine in the presence of primary amines, to produce N(1)-substituted thymines.
In practice, the above techniques are typically performed by an improvised arrangement, using one or more ultraviolet lightbulbs positioned in some convenient location in the laboratory, such as a fumehood or lab benchtop. The DNA specimens (typically on a substrate) would simply be placed an appropriate distance from the UV lights such that the UV flux on the DNA is approximately that desired, and the lights manually energized and de-energized following elapse of an appropriate time.
There are several difficulties associated with the foregoing procedure. First, the extent to which any reaction occurs, for example, cross-linking of DNA to a substrate or formation of thymine dimers, is dependent upon the total energy received by the DNA specimen. This is a function of the UV flux received by the specimen at any given time, over the total time of exposure. However, the flux of a UV light source, typically a low pressure mercury lamp, is not constant over the life of the source. Furthermore, previously typically more than 1 UV lamp was used simultaneously to irradiate the DNA specimen. Should a total or partial failure occur in one of the lamps, the total flux received by the specimen would drop by an unknown quantity. The foregoing factors, which lead to variations in flux received by a DNA specimen either over a given experiment, or from experiment to experiment, lead to a lack of accurately reproducible results. Another difficulty with the previously improvised method, at least in the case where the DNA was to be cross-linked to a substrate, was that often a laboratory technician would at best roughly guess the flux from the UV light source to be used, and then additionally roughly guess the time of exposure required to supply the required total energy (which may be available from a reference, but was typically also estimated). This again led to results which were not accurately reproducible. Furthermore, the typical improvised arrangement, could likely result in UV leakage. The dangers of shortwave UV are well known, and thus prior arrangements could result in hazardous UV exposure to laboratory workers.